PROJECT SUMMARY The human brain is composed of neurons and glia, both of which are derived from multipotent neural progenitor cells (NPCs). Precisely controlled differentiation of multipotent NPCs into neuronal and glial lineages is critical for normal brain development and function, as well as repair of lesions in the central nervous system. Abnormalities arising during this process are thought to underlie the pathogenesis of many neurobiological diseases and neuropsychiatric disorders. Malfunction of the selective RNA binding protein Quaking (QKI) is one such abnormality implicated in neuropsychiatric diseases and myelin disorders in which aberrant neuron-glia development contributes to disease etiology. In neural lineage, QKI expression is detected in rodent NPCs and continues to increase in glia, but is absent in mature neurons. Emerging evidence suggests that silencing of QKI during mouse brain development advances neuronal differentiation. However, whether and how QKI indeed controls human neuron-glia fate selection still remains undefined. Three QKI isoforms with distinct nuclear-cytoplasmic distribution are derived from alternative splicing of C-terminal coding exons. Our preliminary data revealed that cyclin-dependent kinase 5 (CDK5) phosphorylates QKI, which drastically enhances nuclear translocation of cytoplasmic QKI isoforms. Moreover, in a human NPC cell line, CDK5-dependent phosphorylation of QKI regulates the biogenesis of microRNA-124 (miR-124), a key factor that drives neuronal differentiation and inhibits glial lineage in rodents. These data lead to our intriguing hypothesis that the CDK5QKI-miR-124 pathway in human NPCs (hNPCs) may control neuron-glia fate decision and lineage differentiation. In this application, we propose two specific aims to test the aforementioned hypothesis. Aim 1: We will delineate molecular mechanisms by which QKI regulates miR-124 biogenesis in hNPCs. Aim 2: Using CRISPR-Cas9 technology, we will delete the 3? exons that encode individual QKI isoforms in hNPCs and determine the roles of each QKI isoform in hNPC renewal and neuron-glia fate selection. These studies will uncover novel mechanisms that control microRNA-driven neuron-glia lineage development and provide the first evidence demonstrating the function of individual QKI isoforms in hNPCs.